G Model
ARTICLE IN PRESS
ANTAGE 4490 1–3
International Journal of Antimicrobial Agents xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short Communication
1
Tigecycline exhibits inhibitory activity against Clostridium difficile in the intestinal tract of hospitalised patients
2
3
4
Q1
5 6 7
Sirisha Kundrapu a , Kelly Hurless a , Venkata C.K. Sunkesula a , Myreen Tomas a,b , Curtis J. Donskey a,b,∗ a b
Department of Medicine, Infectious Diseases Division, Case Western Reserve University School of Medicine, Cleveland, OH, USA Geriatric Research, Education and Clinical Center, Cleveland Veterans Affairs Medical Center, Cleveland, OH, USA
8
9 19
a r t i c l e
i n f o
a b s t r a c t
10 11 12 13
Article history: Received 30 September 2014 Accepted 18 November 2014
14
18
Keywords: Inhibition Clostridium difficile Tigecycline
20
1. Introduction
15 16 17
21Q2 22 23 24 25 26 27 28 29 30 31 32 33 34 35
In comparison with many other broad-spectrum antibiotics, tigecycline appears to be infrequently associated with Clostridium difficile infection (CDI) [1–3]. Because tigecycline is excreted in significant concentrations in bile and has potent inhibitory activity against C. difficile [1–3], it is plausible that it may achieve sufficient concentrations in the intestinal tract to inhibit colonisation by C. difficile. This hypothesis is supported by a recent small case series in which intravenous tigecycline was an effective adjunctive therapy for refractory CDI [4]. In addition, in mice and in a human gut model, tigecycline inhibited growth and toxin production by C. difficile. Here we tested the hypothesis that tigecycline achieves sufficient concentrations in the intestinal tract to inhibit C. difficile colonisation in patients. A secondary goal of the study was to examine the impact of tigecycline therapy on the indigenous intestinal microbiota, including Bacteroides spp.
36
2. Materials and methods
37
2.1. Study design
38 39
No new acquisition of Clostridium difficile occurred among 12 hospitalised patients receiving tigecycline, and pre-existing colonisation was reduced to undetectable levels in 2 patients. Moreover, 91% of stool suspensions obtained during tigecycline therapy exhibited inhibitory activity against C. difficile. These results suggest that tigecycline achieves sufficient concentrations to inhibit intestinal colonisation by C. difficile. © 2015 Published by Elsevier B.V.
A prospective cohort study of all consenting inpatients receiving tigecycline therapy during a 12-month period was conducted.
∗ Corresponding author. Present address: Geriatric Research, Education and Clinical Center, Cleveland VA Medical Center, 10701 East Boulevard, Cleveland, OH 44106, USA. Tel.: +1 2167913800x4788. E-mail address: [email protected] (C.J. Donskey).
For comparison, control patients from the same wards who were receiving antibiotics with poor in vitro activity against C. difficile, as defined by Owens et al. [5] (i.e. cephalosporins, ciprofloxacin and trimethoprim/sulfamethoxazole), were enrolled. Stool samples were collected before the start of therapy (if possible), on Days 3–5 and 7–10 of therapy, and 5–7 days after completion of therapy. Medical record review was conducted to obtain information on demographics, medical conditions, previous CDI, and antibiotic and proton pump inhibitor use. The Institutional Review Board of Cleveland Veterans Affairs Medical Center (Cleveland, OH) approved the study protocol. 2.2. Microbiological methods Fresh stool specimens were transferred to an anaerobic chamber (Coy Laboratories, Grass Lake, MI). To culture C. difficile, aliquots of stool were plated directly onto pre-reduced C. difficile Brucella Agar containing taurocholic acid and lysozyme (CDBA-TAL) and were incubated for 48 h [6]. Isolates were confirmed to be C. difficile on the basis of typical odour and appearance of colonies and by a positive reaction using C. difficile latex agglutination (Microgen Bioproducts Ltd., Camberley, UK). All C. difficile isolates were tested for in vitro cytotoxin production using C. difficile TOX A/B II (Wampole Laboratories, Princeton, NJ); isolates that did not produce toxin were excluded from the analysis. Concentrations of enterococci, vancomycin-resistant enterococci (VRE) and facultative Gram-negative bacilli were measured using standard quantitative cultures. Minimum inhibitory concentrations (MICs) of tigecycline for C. difficile and VRE isolates recovered before,
http://dx.doi.org/10.1016/j.ijantimicag.2014.11.016 0924-8579/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Kundrapu S, et al. Tigecycline exhibits inhibitory activity against Clostridium difficile in the intestinal tract of hospitalised patients. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.11.016
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G Model ANTAGE 4490 1–3
ARTICLE IN PRESS S. Kundrapu et al. / International Journal of Antimicrobial Agents xxx (2015) xxx–xxx
72
during and after therapy were measured in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [7]. To screen for the emergence of tigecycline-resistant enterococci and facultative Gram-negative bacilli, all stool samples were plated on appropriate selective media containing 2 g/mL tigecycline (Wyeth Pharmaceuticals Inc., Philadelphia, PA).
73
2.3. In vitro assay for inhibitory activity in faecal suspensions
67 68 69 70 71
95
To assess whether inhibitory concentrations of tigecycline were present in stool, a modification of the in vitro assay of colonisation resistance developed by Borriello and Barclay [8] was used. One epidemic NAP1 strain (VA17) and one non-epidemic strain (VA11) were tested (tigecycline MICs ≤ 0.012 g/mL). Fresh stool specimens were homogenised in a 1:1 dilution of pre-reduced sterile water and were inoculated with 104 CFU/mL of vegetative C. difficile inside the anaerobic chamber. Serially diluted specimens were plated onto CDBA plates to determine the concentration of C. difficile immediately after inoculation and after incubation at 37 ◦ C for 24 h. Specimens were excluded if they contained detectable levels of C. difficile prior to the inoculation of vegetative C. difficile or if they were collected during a period when the subject received concurrent therapy with tigecycline and another agent with inhibitory activity against C. difficile [5]. Stool suspensions were considered inhibitory if the concentration of C. difficile decreased or remained unchanged compared with the baseline concentration. Inhibition of the C. difficile isolates was concurrently assessed in sterile filtrates of the stool suspensions. The filtrates were produced by centrifuging the suspensions at 10 000 rpm for 10 min, followed by filtering the supernatant through a 0.22 m filter (Fisher Scientific UK Ltd., Loughborough, UK).
96
2.4. Statistical analysis
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97 98 99 100
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Fisher’s exact test was used to compare the proportions of stool specimens with inhibitory activity against C. difficile. Data were analysed using SPSS statistical software v.10.0 (SPSS Inc., Chicago, IL) and STATA 11 (StataCorp, College Station, TX). 3. Results 3.1. Patient characteristics and frequency of Clostridium difficile colonisation Of 17 patients treated with tigecycline during the study period, 12 were enrolled in the study. The mean age of the 12 subjects was 64 years (range, 44–86 years), 2 (17%) were nursing home residents, 8 (67%) received concurrent antibiotics for part of the course of tigecycline and 3 (25%) had previous CDI within 3 months. Fig. 1 shows the frequency of colonisation with toxigenic C. difficile before, during and after tigecycline treatment. Of five tigecyclinetreated patients with stool specimens available prior to the start of treatment, 2 (40%) were colonised with toxigenic C. difficile. For both of these patients with pre-existing colonisation, tigecycline therapy was associated with a reduction in the concentration of C. difficile to below the limit of detection (1 log10 CFU/g of stool) (Fig. 2, patients 2 and 3). Of nine tigecycline-treated patients with stool specimens available after completion of treatment, 1 (11%) acquired C. difficile colonisation (Fig. 2, patient 1). During tigecycline therapy, none of the 12 patients acquired new colonisation with C. difficile, whereas 4 (33%) of 12 control patients receiving non-inhibitory antibiotics acquired colonisation (P = 0.09). For all C. difficile strains, the tigecycline MIC was ≤0.012 g/mL. The median concentration of tigecycline in stool specimens was 2 g/g (range, 0–6 g/g).
Fig. 1. Frequency of colonisation with Clostridium difficile in 12 study patients before (n = 5 patients cultured), during (n = 12 patients cultured) and after (n = 9 patients cultured) treatment with tigecycline. None of the 12 patients acquired new colonisation with C. difficile during tigecycline therapy.
3.2. In vitro inhibitory activity of faecal suspensions
125
Fig. 3 provides a comparison of the proportions of faecal suspensions before, during and after therapy that were inhibitory to in vitro growth of C. difficile and the mean (±standard error) change in C. difficile concentration for tigecycline versus noninhibitory antibiotics. Suspensions obtained during tigecycline therapy were significantly more likely to be inhibitory than suspensions obtained before or after tigecycline therapy. In addition, suspensions obtained from patients receiving non-inhibitory antibiotics were significantly less likely to be inhibitory than suspensions from patients during tigecycline therapy. For inhibitory faecal suspensions of patients receiving tigecycline, faecal filtrates also consistently inhibited the growth of C. difficile, suggesting that inhibition was due to the presence of tigecycline rather than competing bacteria. In contrast, faecal filtrates produced from inhibitory suspensions of patients receiving non-inhibitory antibiotics did not inhibit growth of C. difficile. Tigecycline therapy resulted in a modest suppression of total enterococci (mean concentration before versus during therapy, 9.2 CFU/g of stool versus 7.8 log10 CFU/g of stool; P = 0.009). Three patients acquired VRE colonisation while on tigecycline and four of five patients with pre-existing VRE colonisation maintained colonisation during therapy. The MICs of VRE strains recovered during tigecycline therapy ranged from 2 g/mL to 8 g/mL. Tigecycline did not suppress total facultative Gram-negative bacilli (mean concentration before versus during therapy, 7.2 log10 CFU/g of stool versus 7.7 log10 CFU/g of stool; P = 0.54); however, only one isolate with elevated MIC to tigecycline (Klebsiella pneumoniae, Patient 1
8
Patient 2
Patient 3
7 Log 10 CFU/g of stool
2
6 5 4 3 2 1 0 Baseline
On treatment
After
Fig. 2. Concentration of Clostridium difficile in the stool of two patients colonised with toxigenic C. difficile at baseline (patients 2 and 3) and the one patient who acquired colonisation after completion of treatment (patient 1).
Please cite this article in press as: Kundrapu S, et al. Tigecycline exhibits inhibitory activity against Clostridium difficile in the intestinal tract of hospitalised patients. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.11.016
126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152
G Model ANTAGE 4490 1–3
ARTICLE IN PRESS S. Kundrapu et al. / International Journal of Antimicrobial Agents xxx (2015) xxx–xxx
3
Fig. 3. In vitro growth of Clostridium difficile in stool suspensions of hospitalised patients treated with tigecycline in comparison with patients treated with antibiotics with minimal in vitro inhibitory activity against C. difficile (i.e. cephalosporins, ciprofloxacin and trimethoprim/sulfamethoxazole). Specimens were considered inhibitory if the concentration of C. difficile decreased or remained unchanged compared with the baseline concentration. SE, standard error.
156
MIC = 8 g/mL) was recovered during tigecycline therapy. Tigecycline therapy did not result in suppression of Bacteroides spp. (mean concentration before versus during therapy, 5.1 CFU/g of stool versus 4.9 log10 CFU/g of stool; P = 0.9).
157
4. Discussion
153 154 155
158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179
Among 12 patients receiving tigecycline, none acquired new colonisation with C. difficile during therapy, and pre-existing colonisation was reduced to undetectable levels in 2 patients. Moreover, 91% of stool suspensions or filtrates obtained during tigecycline therapy exhibited inhibitory activity against C. difficile. These results provide additional evidence that tigecycline achieves sufficient concentrations in the intestinal tract to inhibit colonisation by C. difficile in patients. In addition, these data provide further evidence that tigecycline may have relatively limited impact on the indigenous microbiota of the colon, including Bacteroides spp. Although tigecycline had relatively low MICs for VRE strains recovered from stool, tigecycline therapy did not eliminate preexisting VRE colonisation consistently or prevent new acquisition of colonisation. In addition, tigecycline only modestly reduced total counts of indigenous enterococci. These data may potentially be related to the fact that tigecycline has less potent activity against enterococci than C. difficile and/or by an inoculum effect (i.e. levels of enterococci were very high prior to tigecycline therapy, presumably due to disruption of the microbiota by prior antibiotic therapy). Tigecycline also did not reduce counts of total facultative Gram-negative bacilli. This study has some limitations. The number of subjects studied was small and all of them were male. Although in vitro inhibitory
activity was demonstrated, additional studies including large numbers of treated patients will be needed to evaluate whether the incidence of CDI is reduced in patients receiving tigecycline versus other broad-spectrum antibiotics. Funding: This work was supported by the Department of Veter- Q3 ans Affairs by a grant from Pfizer to CJD. Q4 Competing interests: None declared. Ethical approval: The Institutional Review Board of Cleveland Veterans Affairs Medical Center (Cleveland, OH) approved the study protocol. References [1] Nord CE, Sillerström E, Wahlund E. Effect of tigecycline on normal oropharyngeal and intestinal microflora. Antimicrob Agents Chemother 2006;50: 3375–80. [2] Baines SD, Saxton K, Freeman J, Wilcox MH. Tigecycline does not induce proliferation or cytotoxin production by epidemic Clostridium difficile strains in a human gut model. J Antimicrob Chemother 2006;58:1062–5. [3] Wilcox MH. Evidence for low risk of Clostridium difficile infection associated with tigecycline. Clin Microbiol Infect 2007;13:949–52. [4] Herpers BL, Vlaminckx B, Burkhardt O, Blom H, Biemond-Moeniralam HS, Hornef M, et al. Intravenous tigecycline as adjunctive or alternative therapy for severe refractory Clostridium difficile infection. Clin Infect Dis 2009;48:1732–5. [5] Owens Jr RC, Donskey CJ, Gaynes RP, Loo VG, Muto CA. Antimicrobialassociated risk factors for Clostridium difficile infection. Clin Infect Dis 2008;46: 19–31. [6] Nerandzic MM, Donskey CJ. Effective and reduced-cost modified selective medium for isolation of Clostridium difficile. J Clin Microbiol 2009;47: 397–400. [7] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twentieth informational supplement. Document M100-S20. Wayne, PA: CLSI; 2010. [8] Borriello SP, Barclay FE. An in-vitro model of colonisation resistance to Clostridium difficile infection. J Med Microbiol 1986;21:299–309.
Please cite this article in press as: Kundrapu S, et al. Tigecycline exhibits inhibitory activity against Clostridium difficile in the intestinal tract of hospitalised patients. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.11.016
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ARTICLE IN PRESS
ANTAGE 4490 1–3
International Journal of Antimicrobial Agents xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short Communication
1
Tigecycline exhibits inhibitory activity against Clostridium difficile in the intestinal tract of hospitalised patients
2
3
4
Q1
5 6 7
Sirisha Kundrapu a , Kelly Hurless a , Venkata C.K. Sunkesula a , Myreen Tomas a,b , Curtis J. Donskey a,b,∗ a b
Department of Medicine, Infectious Diseases Division, Case Western Reserve University School of Medicine, Cleveland, OH, USA Geriatric Research, Education and Clinical Center, Cleveland Veterans Affairs Medical Center, Cleveland, OH, USA
8
9 19
a r t i c l e
i n f o
a b s t r a c t
10 11 12 13
Article history: Received 30 September 2014 Accepted 18 November 2014
14
18
Keywords: Inhibition Clostridium difficile Tigecycline
20
1. Introduction
15 16 17
21Q2 22 23 24 25 26 27 28 29 30 31 32 33 34 35
In comparison with many other broad-spectrum antibiotics, tigecycline appears to be infrequently associated with Clostridium difficile infection (CDI) [1–3]. Because tigecycline is excreted in significant concentrations in bile and has potent inhibitory activity against C. difficile [1–3], it is plausible that it may achieve sufficient concentrations in the intestinal tract to inhibit colonisation by C. difficile. This hypothesis is supported by a recent small case series in which intravenous tigecycline was an effective adjunctive therapy for refractory CDI [4]. In addition, in mice and in a human gut model, tigecycline inhibited growth and toxin production by C. difficile. Here we tested the hypothesis that tigecycline achieves sufficient concentrations in the intestinal tract to inhibit C. difficile colonisation in patients. A secondary goal of the study was to examine the impact of tigecycline therapy on the indigenous intestinal microbiota, including Bacteroides spp.
36
2. Materials and methods
37
2.1. Study design
38 39
No new acquisition of Clostridium difficile occurred among 12 hospitalised patients receiving tigecycline, and pre-existing colonisation was reduced to undetectable levels in 2 patients. Moreover, 91% of stool suspensions obtained during tigecycline therapy exhibited inhibitory activity against C. difficile. These results suggest that tigecycline achieves sufficient concentrations to inhibit intestinal colonisation by C. difficile. © 2015 Published by Elsevier B.V.
A prospective cohort study of all consenting inpatients receiving tigecycline therapy during a 12-month period was conducted.
∗ Corresponding author. Present address: Geriatric Research, Education and Clinical Center, Cleveland VA Medical Center, 10701 East Boulevard, Cleveland, OH 44106, USA. Tel.: +1 2167913800x4788. E-mail address: [email protected] (C.J. Donskey).
For comparison, control patients from the same wards who were receiving antibiotics with poor in vitro activity against C. difficile, as defined by Owens et al. [5] (i.e. cephalosporins, ciprofloxacin and trimethoprim/sulfamethoxazole), were enrolled. Stool samples were collected before the start of therapy (if possible), on Days 3–5 and 7–10 of therapy, and 5–7 days after completion of therapy. Medical record review was conducted to obtain information on demographics, medical conditions, previous CDI, and antibiotic and proton pump inhibitor use. The Institutional Review Board of Cleveland Veterans Affairs Medical Center (Cleveland, OH) approved the study protocol. 2.2. Microbiological methods Fresh stool specimens were transferred to an anaerobic chamber (Coy Laboratories, Grass Lake, MI). To culture C. difficile, aliquots of stool were plated directly onto pre-reduced C. difficile Brucella Agar containing taurocholic acid and lysozyme (CDBA-TAL) and were incubated for 48 h [6]. Isolates were confirmed to be C. difficile on the basis of typical odour and appearance of colonies and by a positive reaction using C. difficile latex agglutination (Microgen Bioproducts Ltd., Camberley, UK). All C. difficile isolates were tested for in vitro cytotoxin production using C. difficile TOX A/B II (Wampole Laboratories, Princeton, NJ); isolates that did not produce toxin were excluded from the analysis. Concentrations of enterococci, vancomycin-resistant enterococci (VRE) and facultative Gram-negative bacilli were measured using standard quantitative cultures. Minimum inhibitory concentrations (MICs) of tigecycline for C. difficile and VRE isolates recovered before,
http://dx.doi.org/10.1016/j.ijantimicag.2014.11.016 0924-8579/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Kundrapu S, et al. Tigecycline exhibits inhibitory activity against Clostridium difficile in the intestinal tract of hospitalised patients. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.11.016
40 41 42 43 44 45 46 47 48 49 50
51
52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
G Model ANTAGE 4490 1–3
ARTICLE IN PRESS S. Kundrapu et al. / International Journal of Antimicrobial Agents xxx (2015) xxx–xxx
72
during and after therapy were measured in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [7]. To screen for the emergence of tigecycline-resistant enterococci and facultative Gram-negative bacilli, all stool samples were plated on appropriate selective media containing 2 g/mL tigecycline (Wyeth Pharmaceuticals Inc., Philadelphia, PA).
73
2.3. In vitro assay for inhibitory activity in faecal suspensions
67 68 69 70 71
95
To assess whether inhibitory concentrations of tigecycline were present in stool, a modification of the in vitro assay of colonisation resistance developed by Borriello and Barclay [8] was used. One epidemic NAP1 strain (VA17) and one non-epidemic strain (VA11) were tested (tigecycline MICs ≤ 0.012 g/mL). Fresh stool specimens were homogenised in a 1:1 dilution of pre-reduced sterile water and were inoculated with 104 CFU/mL of vegetative C. difficile inside the anaerobic chamber. Serially diluted specimens were plated onto CDBA plates to determine the concentration of C. difficile immediately after inoculation and after incubation at 37 ◦ C for 24 h. Specimens were excluded if they contained detectable levels of C. difficile prior to the inoculation of vegetative C. difficile or if they were collected during a period when the subject received concurrent therapy with tigecycline and another agent with inhibitory activity against C. difficile [5]. Stool suspensions were considered inhibitory if the concentration of C. difficile decreased or remained unchanged compared with the baseline concentration. Inhibition of the C. difficile isolates was concurrently assessed in sterile filtrates of the stool suspensions. The filtrates were produced by centrifuging the suspensions at 10 000 rpm for 10 min, followed by filtering the supernatant through a 0.22 m filter (Fisher Scientific UK Ltd., Loughborough, UK).
96
2.4. Statistical analysis
74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94
97 98 99 100
101
102 103
104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124
Fisher’s exact test was used to compare the proportions of stool specimens with inhibitory activity against C. difficile. Data were analysed using SPSS statistical software v.10.0 (SPSS Inc., Chicago, IL) and STATA 11 (StataCorp, College Station, TX). 3. Results 3.1. Patient characteristics and frequency of Clostridium difficile colonisation Of 17 patients treated with tigecycline during the study period, 12 were enrolled in the study. The mean age of the 12 subjects was 64 years (range, 44–86 years), 2 (17%) were nursing home residents, 8 (67%) received concurrent antibiotics for part of the course of tigecycline and 3 (25%) had previous CDI within 3 months. Fig. 1 shows the frequency of colonisation with toxigenic C. difficile before, during and after tigecycline treatment. Of five tigecyclinetreated patients with stool specimens available prior to the start of treatment, 2 (40%) were colonised with toxigenic C. difficile. For both of these patients with pre-existing colonisation, tigecycline therapy was associated with a reduction in the concentration of C. difficile to below the limit of detection (1 log10 CFU/g of stool) (Fig. 2, patients 2 and 3). Of nine tigecycline-treated patients with stool specimens available after completion of treatment, 1 (11%) acquired C. difficile colonisation (Fig. 2, patient 1). During tigecycline therapy, none of the 12 patients acquired new colonisation with C. difficile, whereas 4 (33%) of 12 control patients receiving non-inhibitory antibiotics acquired colonisation (P = 0.09). For all C. difficile strains, the tigecycline MIC was ≤0.012 g/mL. The median concentration of tigecycline in stool specimens was 2 g/g (range, 0–6 g/g).
Fig. 1. Frequency of colonisation with Clostridium difficile in 12 study patients before (n = 5 patients cultured), during (n = 12 patients cultured) and after (n = 9 patients cultured) treatment with tigecycline. None of the 12 patients acquired new colonisation with C. difficile during tigecycline therapy.
3.2. In vitro inhibitory activity of faecal suspensions
125
Fig. 3 provides a comparison of the proportions of faecal suspensions before, during and after therapy that were inhibitory to in vitro growth of C. difficile and the mean (±standard error) change in C. difficile concentration for tigecycline versus noninhibitory antibiotics. Suspensions obtained during tigecycline therapy were significantly more likely to be inhibitory than suspensions obtained before or after tigecycline therapy. In addition, suspensions obtained from patients receiving non-inhibitory antibiotics were significantly less likely to be inhibitory than suspensions from patients during tigecycline therapy. For inhibitory faecal suspensions of patients receiving tigecycline, faecal filtrates also consistently inhibited the growth of C. difficile, suggesting that inhibition was due to the presence of tigecycline rather than competing bacteria. In contrast, faecal filtrates produced from inhibitory suspensions of patients receiving non-inhibitory antibiotics did not inhibit growth of C. difficile. Tigecycline therapy resulted in a modest suppression of total enterococci (mean concentration before versus during therapy, 9.2 CFU/g of stool versus 7.8 log10 CFU/g of stool; P = 0.009). Three patients acquired VRE colonisation while on tigecycline and four of five patients with pre-existing VRE colonisation maintained colonisation during therapy. The MICs of VRE strains recovered during tigecycline therapy ranged from 2 g/mL to 8 g/mL. Tigecycline did not suppress total facultative Gram-negative bacilli (mean concentration before versus during therapy, 7.2 log10 CFU/g of stool versus 7.7 log10 CFU/g of stool; P = 0.54); however, only one isolate with elevated MIC to tigecycline (Klebsiella pneumoniae, Patient 1
8
Patient 2
Patient 3
7 Log 10 CFU/g of stool
2
6 5 4 3 2 1 0 Baseline
On treatment
After
Fig. 2. Concentration of Clostridium difficile in the stool of two patients colonised with toxigenic C. difficile at baseline (patients 2 and 3) and the one patient who acquired colonisation after completion of treatment (patient 1).
Please cite this article in press as: Kundrapu S, et al. Tigecycline exhibits inhibitory activity against Clostridium difficile in the intestinal tract of hospitalised patients. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.11.016
126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152
G Model ANTAGE 4490 1–3
ARTICLE IN PRESS S. Kundrapu et al. / International Journal of Antimicrobial Agents xxx (2015) xxx–xxx
3
Fig. 3. In vitro growth of Clostridium difficile in stool suspensions of hospitalised patients treated with tigecycline in comparison with patients treated with antibiotics with minimal in vitro inhibitory activity against C. difficile (i.e. cephalosporins, ciprofloxacin and trimethoprim/sulfamethoxazole). Specimens were considered inhibitory if the concentration of C. difficile decreased or remained unchanged compared with the baseline concentration. SE, standard error.
156
MIC = 8 g/mL) was recovered during tigecycline therapy. Tigecycline therapy did not result in suppression of Bacteroides spp. (mean concentration before versus during therapy, 5.1 CFU/g of stool versus 4.9 log10 CFU/g of stool; P = 0.9).
157
4. Discussion
153 154 155
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Among 12 patients receiving tigecycline, none acquired new colonisation with C. difficile during therapy, and pre-existing colonisation was reduced to undetectable levels in 2 patients. Moreover, 91% of stool suspensions or filtrates obtained during tigecycline therapy exhibited inhibitory activity against C. difficile. These results provide additional evidence that tigecycline achieves sufficient concentrations in the intestinal tract to inhibit colonisation by C. difficile in patients. In addition, these data provide further evidence that tigecycline may have relatively limited impact on the indigenous microbiota of the colon, including Bacteroides spp. Although tigecycline had relatively low MICs for VRE strains recovered from stool, tigecycline therapy did not eliminate preexisting VRE colonisation consistently or prevent new acquisition of colonisation. In addition, tigecycline only modestly reduced total counts of indigenous enterococci. These data may potentially be related to the fact that tigecycline has less potent activity against enterococci than C. difficile and/or by an inoculum effect (i.e. levels of enterococci were very high prior to tigecycline therapy, presumably due to disruption of the microbiota by prior antibiotic therapy). Tigecycline also did not reduce counts of total facultative Gram-negative bacilli. This study has some limitations. The number of subjects studied was small and all of them were male. Although in vitro inhibitory
activity was demonstrated, additional studies including large numbers of treated patients will be needed to evaluate whether the incidence of CDI is reduced in patients receiving tigecycline versus other broad-spectrum antibiotics. Funding: This work was supported by the Department of Veter- Q3 ans Affairs by a grant from Pfizer to CJD. Q4 Competing interests: None declared. Ethical approval: The Institutional Review Board of Cleveland Veterans Affairs Medical Center (Cleveland, OH) approved the study protocol. References [1] Nord CE, Sillerström E, Wahlund E. Effect of tigecycline on normal oropharyngeal and intestinal microflora. Antimicrob Agents Chemother 2006;50: 3375–80. [2] Baines SD, Saxton K, Freeman J, Wilcox MH. Tigecycline does not induce proliferation or cytotoxin production by epidemic Clostridium difficile strains in a human gut model. J Antimicrob Chemother 2006;58:1062–5. [3] Wilcox MH. Evidence for low risk of Clostridium difficile infection associated with tigecycline. Clin Microbiol Infect 2007;13:949–52. [4] Herpers BL, Vlaminckx B, Burkhardt O, Blom H, Biemond-Moeniralam HS, Hornef M, et al. Intravenous tigecycline as adjunctive or alternative therapy for severe refractory Clostridium difficile infection. Clin Infect Dis 2009;48:1732–5. [5] Owens Jr RC, Donskey CJ, Gaynes RP, Loo VG, Muto CA. Antimicrobialassociated risk factors for Clostridium difficile infection. Clin Infect Dis 2008;46: 19–31. [6] Nerandzic MM, Donskey CJ. Effective and reduced-cost modified selective medium for isolation of Clostridium difficile. J Clin Microbiol 2009;47: 397–400. [7] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twentieth informational supplement. Document M100-S20. Wayne, PA: CLSI; 2010. [8] Borriello SP, Barclay FE. An in-vitro model of colonisation resistance to Clostridium difficile infection. J Med Microbiol 1986;21:299–309.
Please cite this article in press as: Kundrapu S, et al. Tigecycline exhibits inhibitory activity against Clostridium difficile in the intestinal tract of hospitalised patients. Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2014.11.016
180 181 182 183 184 185 186 187 188 189
190
191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212
